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Artificial polypeptide LH, antibody thereof and application in pathological detection

An artificial and pathological technology, applied in the fields of biological detection and immunology, can solve the problems of no obvious reduction in polymer steric hindrance, no significant increase in the number of antibodies, and many toxic and harmful chemical reagents. Good reproducibility and reproducibility, and the effect of improving sensitivity and specificity

Active Publication Date: 2021-04-23
HANGZHOU BIOLYNX TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(4) The secondary antibody developed by Roche Diagnostic Reagents Co., Ltd. is a combination of naturally occurring small molecule haptens (such as pesticides, veterinary drugs, antibiotics, quantum dots, etc.) Polymer anti-small molecule hapten antibody combination, so as to realize the immunohistochemical detection of histone; however, this method requires natural hapten small molecules, and the cross-linking process requires more toxic and harmful chemical reagents, and the coupling process is complicated, so the production cost higher
[0004] At present, there is no major improvement in polymer structure design and preparation technology in the existing technology, which is embodied in: the number of enzymes and antibodies per unit volume does not increase significantly; The steric hindrance of the object has not been significantly reduced, resulting in no improvement in sensitivity, so there is an urgent need for enhanced secondary antibodies for pathological detection with better effects

Method used

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  • Artificial polypeptide LH, antibody thereof and application in pathological detection
  • Artificial polypeptide LH, antibody thereof and application in pathological detection
  • Artificial polypeptide LH, antibody thereof and application in pathological detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of Enhanced Secondary Antibody System Using Artificial Peptides

[0069] This example provides a method for preparing an enhanced secondary antibody using an artificial polypeptide and its application in pathological detection.

[0070] First, artificial polypeptides are prepared. The artificial polypeptide sequences are completely artificially designed and synthesized. The design principle is: the sequences that do not naturally exist in mammals, and the antibodies produced by them have no specific cross-reaction with mammals, especially with proteins on human tissues. .

[0071] Secondly, prepare a polyclonal antibody against the primary antibody and couple it with an artificial polypeptide to form an artificial polypeptide-antibody conjugate, that is, the post-primary antibody reagent. A polyclonal antibody can be conjugated to multiple artificial polypeptides to amplify the detection signal.

[0072] Then, prepare the anti-artificial polypept...

Embodiment 2

[0084] Embodiment 2 artificial polypeptide

[0085] The artificial polypeptide involved in this embodiment refers to a polypeptide that does not naturally exist in mammals, especially in human tissues.

[0086] Artificial polypeptide PL: the amino acid sequence is TTT(PTT)n, n=1~10, namely:

[0087] TTTPTT (SEQ ID NO: 1),

[0088] TTTPTTPTT (SEQ ID NO: 2),

[0089] TTTPTTPTTPTT (SEQ ID NO: 3),

[0090] TTTPTTPTTPTTPTT (SEQ ID NO: 4),

[0091] TTTPTTPTTPTTPTTPTT (SEQ ID NO: 5),

[0092] TTTPTTPTTPTTPTTPTTPTT (SEQ ID NO: 6),

[0093] TTTPTTPTTPTTPTTPTTPTTPTT (SEQ ID NO: 7),

[0094] TTTPTTPTTPTTPTTPTTPTTPTTPTT (SEQ ID NO: 8),

[0095] TTTPTTPTTPTTPTTPTTPTPTTPTTPTTPTT (SEQ ID NO: 9), or

[0096] TTTPTTPTTPTTPTTPTTPTTPTTPTTPTTPTT (SEQ ID NO: 10),

[0097] In order to facilitate protein coupling, a cysteine ​​(C) can be added at the N-terminal or C-terminal of this amino acid.

[0098] Artificial polypeptide HM: the amino acid sequence is AAP(AADAAD)nAAPAAA, n=1~10, that ...

Embodiment 3

[0122] Embodiment 3 Preparation of Anti-artificial Polypeptide Antibody

[0123] Rabbits were immunized with the artificial polypeptide PL (TTTPTTPTT) in Example 2 to further prepare rabbit-derived monoclonal antibodies. The antibody can specifically bind to the artificial polypeptide PL, and the ELISA binding curve is shown in figure 2 , EC50 was 9.94ng / mL.

[0124] The anti-artificial polypeptide PL antibody was analyzed using Kabat online software, and the results showed that the variable region sequence of the antibody had the following information:

[0125] (1) The amino acid sequence of CDR-H1 includes: TNAMS (SEQ ID NO: 31).

[0126] (2) The amino acid sequence of CDR-H2 includes: IISSSGSTYYARWAKG (SEQ ID NO: 32).

[0127] (3) The amino acid sequence of CDR-H3 includes: GNI (SEQ ID NO: 33).

[0128] (4) The amino acid sequence of CDR-L1 includes: QSSQSVYNNNLA (SEQ ID NO: 34).

[0129] (5) CDR-L2 amino acid sequence includes: RASKLAS (SEQ ID NO: 35).

[0130] (6) ...

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PUM

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Abstract

The invention discloses an artificial polypeptide LH, an antibody thereof and an application in pathological detection, and belongs to the technical field of biological detection and the field of immunology. The polypeptide LH has an amino acid sequence shown as any one of SEQ ID NO: 21-30, and heavy chain variable regions CDR-H1, CDR-H2 and CDR-H3 and light chain variable regions CDR-L1, CDR-L2 and CDR-L3 of a monoclonal antibody of the artificial polypeptide LH have amino acid sequences shown as SEQ ID NO: 43-48 respectively. The artificial polypeptide LH and the antibody thereof are used for establishing a pathology enhanced secondary antibody detection method. The method has the advantages of high stability and good repeatability, and is especially suitable for detection of proteins with low expression abundance. The specificity is good, the sensitivity is high, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of biological detection and the field of immunology, specifically, an artificial polypeptide LH, its antibody and its application in pathological detection. Background technique [0002] Immunohistochemistry (IHC) refers to the qualitative, localization or quantitative determination of the corresponding antigen in the pathological tissue through the antigen-antibody reaction and histochemical color reaction of the specific antibody labeled with the chromogenic agent in situ on the tissue cells. An immunological detection technique. The principle of immunohistochemical detection is: the tissue section is incubated with the primary antibody reagent after antigen heat repair, and the antigen-antibody complex of the primary antibody and the target antigen is formed in situ; the primary antibody molecule in the antigen-antibody complex is then combined with the enzyme The labeled polymer secondary antibody is bo...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/00C07K7/06C07K16/44C07K19/00G01N33/569G01N33/577G01N33/58
CPCC07K7/08C07K14/001C07K7/06C07K16/44G01N33/56966G01N33/577G01N33/581C07K2317/565C07K2317/92C07K2319/00
Inventor 潘丽李明振蔡宁
Owner HANGZHOU BIOLYNX TECH CO LTD